114 research outputs found
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Repurposing the GNAT Fold in the Initiation of Polyketide Biosynthesis.
Natural product biosynthetic pathways are replete with enzymes repurposed for new catalytic functions. In some modular polyketide synthase (PKS) pathways, a GCN5-related N-acetyltransferase (GNAT)-like enzyme with an additional decarboxylation function initiates biosynthesis. Here, we probe two PKS GNAT-like domains for the dual activities of S-acyl transfer from coenzyme A (CoA) to an acyl carrier protein (ACP) and decarboxylation. The GphF and CurA GNAT-like domains selectively decarboxylate substrates that yield the anticipated pathway starter units. The GphF enzyme lacks detectable acyl transfer activity, and a crystal structure with an isobutyryl-CoA product analog reveals a partially occluded acyltransfer acceptor site. Further analysis indicates that the CurA GNAT-like domain also catalyzes only decarboxylation, and the initial acyl transfer is catalyzed by an unidentified enzyme. Thus, PKS GNAT-like domains are re-classified as GNAT-like decarboxylases. Two other decarboxylases, malonyl-CoA decarboxylase and EryM, reside on distant nodes of the superfamily, illustrating the adaptability of the GNAT fold
Determining crystal structures through crowdsourcing and coursework
We show here that computer game players can build high-quality crystal structures. Introduction of a new feature into the computer game Foldit allows players to build and real-space refine structures into electron density maps. To assess the usefulness of this feature, we held a crystallographic model-building competition between trained crystallographers, undergraduate students, Foldit players and automatic model-building algorithms. After removal of disordered residues, a team of Foldit players achieved the most accurate structure. Analysing the target protein of the competition, YPL067C, uncovered a new family of histidine triad proteins apparently involved in the prevention of amyloid toxicity. From this study, we conclude that crystallographers can utilize crowdsourcing to interpret electron density information and to produce structure solutions of the highest quality
A Mononuclear Iron-Dependent Methyltransferase Catalyzes Initial Steps in Assembly of the Apratoxin A Polyketide Starter Unit.
Domain Organization and Active Site Architecture of a Polyketide Synthase Cmethyltransferase
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Biosynthesis of t‑Butyl in Apratoxin A: Functional Analysis and Architecture of a PKS Loading Module
The unusual feature of a t-butyl group is found in several marine-derived natural products including apratoxin A, a Sec61 inhibitor produced by the cyanobacterium Moorea bouillonii PNG 5-198. Here, we determine that the apratoxin A t-butyl group is formed as a pivaloyl acyl carrier protein (ACP) by AprA, the polyketide synthase (PKS) loading module of the apratoxin A biosynthetic pathway. AprA contains an inactive "pseudo" GCN5-related N-acetyltransferase domain (ΨGNAT) flanked by two methyltransferase domains (MT1 and MT2) that differ distinctly in sequence. Structural, biochemical, and precursor incorporation studies reveal that MT2 catalyzes unusually coupled decarboxylation and methylation reactions to transform dimethylmalonyl-ACP, the product of MT1, to pivaloyl-ACP. Further, pivaloyl-ACP synthesis is primed by the fatty acid synthase malonyl acyltransferase (FabD), which compensates for the ΨGNAT and provides the initial acyl-transfer step to form AprA malonyl-ACP. Additionally, images of AprA from negative stain electron microscopy reveal multiple conformations that may facilitate the individual catalytic steps of the multienzyme module
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A Mononuclear Iron-Dependent Methyltransferase Catalyzes Initial Steps in Assembly of the Apratoxin A Polyketide Starter Unit
Natural
product biosynthetic pathways contain a plethora of enzymatic
tools to carry out difficult biosynthetic transformations. Here, we
discover an unusual mononuclear iron-dependent methyltransferase that
acts in the initiation steps of apratoxin A biosynthesis (AprA MT1).
Fe<sup>3+</sup>-replete AprA MT1 catalyzes one or two methyl transfer
reactions on the substrate malonyl-ACP (acyl carrier protein), whereas
Co<sup>2+</sup>, Fe<sup>2+</sup>, Mn<sup>2+</sup>, and Ni<sup>2+</sup> support only a single methyl transfer. MT1 homologues exist within
the “GNAT” (GCN5-related <i>N</i>-acetyltransferase)
loading modules of several modular biosynthetic pathways with propionyl,
isobutyryl, or pivaloyl starter units. GNAT domains are thought to
catalyze decarboxylation of malonyl-CoA and acetyl transfer to a carrier
protein. In AprA, the GNAT domain lacks both decarboxylation and acyl
transfer activity. A crystal structure of the AprA MT1-GNAT di-domain
with bound Mn<sup>2+</sup>, malonate, and the methyl donor <i>S</i>-adenosylmethionine (SAM) reveals that the malonyl substrate
is a bidentate metal ligand, indicating that the metal acts as a Lewis
acid to promote methylation of the malonyl α-carbon. The GNAT
domain is truncated relative to functional homologues. These results
afford an expanded understanding of MT1-GNAT structure and activity
and permit the functional annotation of homologous GNAT loading modules
both with and without methyltransferases, additionally revealing their
rapid evolutionary adaptation in different biosynthetic contexts
Domain Organization and Active Site Architecture of a Polyketide Synthase <i>C</i>‑methyltransferase
Polyketide metabolites produced by
modular type I polyketide synthases
(PKS) acquire their chemical diversity through the variety of catalytic
domains within modules of the pathway. Methyltransferases are among
the least characterized of the catalytic domains common to PKS systems.
We determined the domain boundaries and characterized the activity
of a PKS <i>C</i>-methyltransferase (<i>C</i>-MT)
from the curacin A biosynthetic pathway. The <i>C</i>-MT
catalyzes <i>S</i>-adenosylmethionine-dependent methyl transfer
to the α-position of β-ketoacyl substrates linked to acyl
carrier protein (ACP) or a small-molecule analog but does not act
on β-hydroxyacyl substrates or malonyl-ACP. Key catalytic residues
conserved in both bacterial and fungal PKS <i>C</i>-MTs
were identified in a 2 Å crystal structure and validated biochemically.
Analysis of the structure and the sequences bordering the <i>C</i>-MT provides insight into the positioning of this domain
within complete PKS modules
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Structural Basis of Polyketide Synthase O‑Methylation
Modular type I polyketide synthases (PKSs) produce some of the most chemically complex metabolites in nature through a series of multienzyme modules. Each module contains a variety of catalytic domains to selectively tailor the growing molecule. PKS O-methyltransferases ( O-MTs) are predicted to methylate β-hydroxyl or β-keto groups, but their activity and structure have not been reported. We determined the domain boundaries and characterized the catalytic activity and structure of the StiD and StiE O-MTs, which methylate opposite β-hydroxyl stereocenters in the myxobacterial stigmatellin biosynthetic pathway. Substrate stereospecificity was demonstrated for the StiD O-MT. Key catalytic residues were identified in the crystal structures and investigated in StiE O-MT via site-directed mutagenesis and further validated with the cyanobacterial CurL O-MT from the curacin biosynthetic pathway. Initial structural and biochemical analysis of PKS O-MTs supplies a new chemoenzymatic tool, with the unique ability to selectively modify hydroxyl groups during polyketide biosynthesis
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